Fig. 5: Measurements of Ca²⁺ signals at MCSs by PRINCESS.

a Representative images of the 475/390 nm ratio (R, see Methods) of OMM-Cepia3, co-expressed in HeLa cells with either short or long ER-mit RspA-splitFAST to mark short or long ER-mit MCSs. The ratio images of OMM-Cepia3 and of the portion of OMM-Cepia3 co-localized with ER-mit RspA-splitFAST (ER-mit MCSs Cepia3 Ratio) are shown, before (basal) and upon histamine (100 µM) stimulation. On the right, traces represent the ratios of OMM-Cepia3 (OMM) or of the portion of OMM-Cepia3 co-localized with short ER-mit RspA-splitFAST (ER-mit) upon histamine stimulation (arrows), for the three cells on the left. The box plots represent the ΔR of OMM-Cepia3 upon histamine stimulation either in the bulk OMM or in the regions co-localized with ER-mit MCSs, in cells expressing either short or long ER-mit RspA-splitFAST, as indicated. b OMM-Cepia3, expressed in HeLa cells, was calibrated by permeabilizing cells with digitonin (25 µM) in intracellular-like buffer and adding fixed Ca²⁺ concentrations. Ratiometric 475/390 nm measurements were performed (see Methods). Mean ± SEM. c The heat map represents the OMM-Cepia3 Ratio over time in the sub-regions co-localized with short ER-mit MCSs (identified as single objects, y axis) of a HeLa cell (as in a). Upon histamine stimulation, ~50% of ER-mit MCSs experiences high Ca²⁺ concentrations (box plot on the right). d The box plot represents the 475/390 nm ratio (R, see Methods) of OMM-GCaMP6f in HeLa cells in basal conditions, either in the bulk OMM or at short ER-mit MCSs (identified by co-expression and co-localization with ER-mit RspA-splitFAST). e The cartoon represents the rationale behind ER-mit PRINCESS design. Mutated Calmodulin (CaM*) from Cepia3 and the M13 peptide were incorporated within ER-mit splitFAST, to provide Ca²⁺-sensing capabilities. Created in BioRender. Garcia Casas, P. (2023) BioRender.com/f06c444. f Representative traces of ER-mit PRINCESS or OMM-PRINCESS fluorescence in HeLa cells, upon histamine (100 µM) stimulation in Ca²⁺-free mKRB (see Methods), or CaCl₂ (2 mM) addition (SOCE) after 6 min depletion of ER Ca²⁺ content (obtained by histamine and thapsigargin (100 nM) stimulation in Ca²⁺-free mKRB). On the right, box plots represent the peaks of OMM- or ER-mit PRINCESS fluorescence (expressed as F/F0) upon the indicated treatments. g Representative confocal images of HeLa cells co-expressing OMM-RFP (used to normalize the fluorescent signal) with either ER-mit PRINCESS or OMM-PRINCESS, before (basal) or upon histamine (100 µM) stimulation in Ca²⁺-free mKRB. The PRINCESS/OMM-RFP ratio images are also shown. See also the corresponding Supplementary Movies 13 and 14. On the right, the corresponding traces (mean ± SEM) of the ratio signals for the indicated conditions are shown. Scale bar: 10 µm (a, g).